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Limb development in Drosophila
by Dr. William Brook
Department of Medical Biochemistry
University of Calgary
In Drosophila, limb development presents
different problems for the control of growth and cell-patterning than embryonic
development. Most of the patterning of the major embryonic axes during early
Drosophila development takes place in the pre-blastoderm embryo.
In this syncytial environment patterning is controlled by concentration
gradients established by the diffusion of maternally localized transcription
factors and RNA binding proteins. Limb are cellular, so different mechanisms
are required to mediate limb development. However, the specification of
different cellular fates is still controlled by gradients of regulatory
proteins, in the case of the limb, the proteins are secreted signaling molecules
instead of transcription factors.
Limbs develop from imaginal discs
limbs (legs, wings, halteres, antennae, mouth parts) derive from structures
called imaginal discs. Imaginal discs begin as small clusters of cells which
are set aside during embryogenesis. These cells proliferate during larval
development to form folded, single layer, epithelial sacs. Each imaginal
disc gives rise to a separate structure so there is a disc for each leg,
wing, haltere and antenna. The cells in the disc cease dividing just prior
to differentiation which begins at the time of pupation. As the discs begin
to differentiate, they evert (or unfold) and fuse to form a continuous adult
head and thoracic cuticle.
(See Browder et al., 1991, Figure 14.8;
Gilbert, 1997, Fig. 19.14; Kalthoff, 1996, Fig. 6.20; Wolpert et al.,
1998, Fig. 2.34)
Imaginal discs are segmentally determined
Imaginal disc cells are specified as to which segmental
structure they will form as early as the cellular blastoderm stage. In fact,
assignment of segmental fate by the homeotic genes occurs before cells are
determined to become either imaginal or larval cells. The segmental determination
is very stable. Classic experiments by Ernst Hadorn involving serial transplantations
of imaginal discs showed that the segmental determination of discs is stable
over many cells generations. In fact, changes in determined states happened
only infrequently and in very consistent patterns. These changes of fate
during growth in culture are referred to as transdetermination. Some
of the changes are likely to be due to changes in the expression of the
(See Browder et al., 1991, Figure 14.9
and 14.10; Kalthoff, 1996, Figs. 6.21-6.25)
Establishment of discs
The cells giving rise to the thoracic imaginal
disc primordia are initially part of the embryonic ectoderm. The primordia
are established straddling the parasegmental boundaries in each of the three
thoracic segments. They are specified in response to two different secreted
signals: wingless (wg), a segment polarity gene and member of the
Wnt family of secreted factors and decapentaplegic (dpp) a Drosophila
TGF-beta homologue. wg is expressed as a stripe just anterior
to the parasegmental boundary. dpp is expressed in a lateral stripe
running perpendicular to the cells expressing wg. The cells in the
vicinity of the intersection between the wg and dpp stripes are exposed
to both secreted signals and become specified as imaginal disc cells. This
was demonstrated by correlating the spatial pattern of genes expressed in
imaginal discs (such as the gene Distal-less) with the expression
patterns of wg and dpp .The effects of wg and dpp
mutations on the formation of imaginal discs and the expression of disc
specific genes were also assessed.
Figure 1 (After Cohen,
Spatial patterning of the anterior-posterior
axis of the wing
(Please refer to Blair, 1995; Brook et al., 1996)
for reviews of the following material.
For the sake of simplicity, we shall consider how cells become specified
as to their position in the anterior-posterior (AP) axis of the wing imaginal
disc as an example of the kinds of mechanisms that control imaginal disc
development. The wing disc primordia begin as small clusters of ~40 cells.
By the time the discs are ready to differentiate the cells in the disc number
approximately 50 000. The complex patterns of cellular differentiation seen
in the wing are not determined in the cells of the primordia, but rather
the patterns of growth and differentiation are specified during the course
of disc development.
The wing is divided along the anterior posterior axis into a series of longitudinal
veins and intervein regions. Each vein has characteristic sense organs and
other structures that indicate that they are distinct from one another.
Intervein regions also have characteristic patterns of differentiation.
So the problem is to understand how these differences become specified during
Figure 2. (Modified from Brook et al.,
Anterior versus Posterior Determination
Figure 3. engrailed expression in
a wing disc (left) and an adult wing(r)
(Modified from Brook et al., 1996)
The first decision cells make during wing development
is whether they are anterior or posterior. The disc primordia are established
straddling the parasegmental border. Cells in the posterior half express
the homeodomain gene engrailed (en), one of the segment polarity
genes. Cells in the two halves of the disc are already determined as anterior
or posterior at the time of disc formation. Cells from the posterior part
never make anterior structures, anterior cells never make posterior structures.
This decision is controlled by whether or not wing cells express the engrailed
gene. How was this shown? The first clues that engrailed controlled
anterior versus posterior fate came from the phenotype of en1 mutant
flies. en1 is a regulatory mutation in the engrailed gene
that results in reduced engrailed expression in the wings. In en1
mutant flies, the posterior part of the wing (which expresses engrailed)
is transformed so that it develops as an anterior wing. The result is that
wing that has a symmetric double anterior pattern.
One difficulty with the interpreting the results of the engrailed
flies is that en1 is not a complete deletion of engrailed
function. Ideally, we would like to know the effects of complete loss of
function for genes when we are studying developmental processes. A problem
that arises when studying limb development is that most of the important
genes cause very early lethality when mutated. In order to circumvent this
problem we must use genetic mosaics. In Drosophila the most common
way of making genetic mosaics is to use somatic mitotic crossing over. This
technique uses irradiation or site specific recombinases to induce mitotic
crossovers between sister chromosomes in somatic cells. This results in
patches of homozygous mutant tissue (i.e. en-/en- or A/A in diagram
below) in heterozygous animals (en-/en+, A/+ in diagram). It is possible
to simultaneously mark the clones with an innocuous genetic marker in order
to distinguish homozygous mutant tissue from the heterozygous background.
Furthermore, the time of induction of clones can also be controlled so the
effects of loss of function for a particular gene at different developmental
stages can be assessed.
Figure 4. Exchange
between homologous chromosomes heterozygous for a mutation (A) and a cell
marker (m) leads to the production of a daughter cell homozygous for both
the mutant and the marker (A m/A m) and a twin homozygous for both wild-type
alleles (+ +/+ +). The homozygous mutant cell will proliferate to produce
a patch of mutant tissue surrounded by wild-type cells. (Modified from Brook
et al., 1996)
When Tetsuya Tabata made clones of cells that lacked
all engrailed function, he noticed two things (Tabata et al.,
1995). First as expected, the mutant cells became anterior in character
if the clone was in the posterior part of the wing (i.e. the engrailed
expressing region) and the clones had no effect if they were located in
the anterior compartment. The second thing he noticed was very surprising.
He found that whenever a clone was induced in the posterior part of the
wing and caused a new confrontation of anterior and posterior cells, the
new confrontation induced the development of a secondary A/P axis in the
wing. This suggested that in normal development, the boundary between cells
expressing engrailed and cells not expressing engrailed acted
as an organizing centre controlling the specification of the anterior-posterior
Figure 5. (Modified from Brook et al.,
1996; see also figures 3 and 6 from Tabata et al., 1995).
What could be mediating this interaction between
the anterior and posterior cells? From studies of segment polarity genes,
it was known that the protein hedgehog was secreted by posterior
cells in the embryonic segment and was responsible for signaling to anterior
cells. hedgehog was also expressed in the posterior cells in the
wing. Konrad Basler and Gary Struhl did a beautiful experiment to show that
the interaction between A and P was mediated by hedgehog. They developed
a technique that allowed the generation of a different kind of genetic mosaic.
This technique (called the flip-out cassette) allowed the production of
patches of tissue, which constitutively expressed hedgehog (or any
other gene). These clones are the reciprocal of the clones induced by somatic
cross-over as they lead to the activation rather than the loss of the gene's
function in clones of cells.
Figure 6. Scheme for producing clones of
cells expressing a gene of interest. The first transgene carries a cell
marker flanked by FRT sites separating a constitutive promoter and a protein
coding sequence of interest (e.g. hh or dpp cDNA). Excision
of the "flip-out" cassette catalyzed by the yeast FLP recombinase,
provided on a second transgene. The flp recombinase is under the control
of the heat-shock promoter and recognizes the FRT sites as targets for site
specific recombination. Production of the flip recombinase under heat shock
control allows the induction of clones expressing the gene of interest at
any stage of development and in any cell in the imaginal disc. The result
is a clone of cells expressing the gene of interest and not the cell-marker
surrounded by non-expressing, marked cells.
(After Basler and Struhl, 1994; modified from Brook et al., 1996)
Basler and Struhl found that clones of cells expressing
hedgehog had no effect in the posterior half of the wing (as expected
because hedgehog is normally expressed there), but cells located in the
anterior half were able to re-organize pattern in a manner similar to the
effects of en mutants clones (Basler and Struhl, 1994). The duplications
produced by the hh clones were symmetric and organized around the cells
expressing hedgehog. This suggested that hedgehog could induce
anterior cells to organize a new A/P axis.
Figure 7. (Modified from Brook et al.,
Organizing the Anterior-Posterior axis
Hedgehog does not organize the anterior posterior
axis directly but rather it induces a second gene at the interface between
the anterior and posterior cell populations. This gene, dpp, is expressed
in the anterior cells in response to the hedgehog signal. If the
clones of cells receiving the hedgehog signal are prevented from
producing dpp, they do not produce axis duplications, indicating
that dpp expression is necessary for hedgehog induced axis duplications.
dpp (remember, it is a secreted protein of the TGF-beta family) is
expressed in a stripe of cells that bisects the wing imaginal disc into
anterior and posterior halves. Its spatial expression pattern and its function
as a signaling molecule made it an excellent candidate to organize the pattern
in the a/p axis. Basler and Struhl were able to show this by making clones
of cells which constitutively expressed dpp. These clones were able
to cause pattern duplications in both the anterior and posterior halves
of the wing (see Figure 5 of Zecca et al., 1995).
Figure 8. (Modified
from Brook et al., 1996)
The results suggested that the way the anterior
posterior axis was organized could be broken down into three steps.
- interaction between anterior and posterior cells
(i.e.. engrailed expressing and non-expressing cells)
- short range signaling from posterior to anterior
- signaling from dpp to control pattern in both
the anterior and posterior halves of the wing.
Direct, long range action of dpp
So, the fate of cells along the A/P axis of the
wing is specified by two factors. The expression of the engrailed gene determines
the anterior versus posterior fate and the distance from the source of dpp
signaling determines what part of the pattern of cell will make. For example,
cells that are posterior (express engrailed) and close to the source will
become vein 4 cells and posterior cells that are farther away become vein
5. Similarly, nearby anterior cells will become vein 3 and anterior cells
that are further away become vein 2. But, the AP axis of the wing is approximately
50 cells in diameter. How is it that dpp influences the development
of cells that are so distant from where it is expressed?
There are two likely explanations. The first is that dpp could diffuse
a short distance and induce a second signal. This signal would induce another
signal which in turn could induce a third signal, etc. This series of cell
interactions could specify different fates in the A/P axis. This is termed
serial induction or signal relay. The second model, direct
long-range action, suggests that dpp diffuses over a long range and
forms a concentration gradient. In this model, cells differentiate into
different structures depending on the concentration of dpp to which they
In order to test these models, it is necessary to manipulate the ability
of cells to receive the dpp signal and observe the effect on cell
fate and molecular marker expression.
Independent signaling molecules organize the A/P
and D/V axes.
The dorsal-ventral axis of the wing is organized
by a similar mechanisms. The interaction between dorsal cells expressing
the homeodomain protein apterous and ventral non-expressing cells
results in the expression of wingless at the interface between the
two cell populations. wingless is able to influence cell fate throughout
the entire wing. The dorsal versus ventral decision is controlled by apterous
in a manner similar to the way engrailed controls anterior versus
Drosophila limbs are derived from embryonic epithelial
sacs called imaginal discs
The cells of the imaginal disc are stabley determined to give rise to a
specific segmental appendage as shown by in vivo culture experiments. Under
these conditions disc cells only rarely change their state of determination
though a process termed transdetermination
The thoracic imaginal disc primordia are set aside early in embryogenesis
in response to the secreted signals wingless and decapentaplegic.
The genetic pathway for the development of the anterior posterior axis involves
i) anterior versus posterior fate determination by the engrailed gene; ii)
posterior to anterior cell signaling mediated by hedgehog that results
in the expression of dpp bisecting the A/P axis; and iii) organization
of the A/P axis by dpp in a direct and concentration dependent manner.
A similar pathway controls the development of the D/V axis.
- What is meant by the transdetermination of imaginal
- Describe the signals that specify ectodermal
cells to become imaginal disc cells. What prevents abdominal cells that
receive wg and dpp from becoming imaginal discs?
- Describe the methods for making clones using
somatic recombination and the "flip-out" technique.
- Describe the role of each of the following genes
in the development of the A/P axis of the wing: engrailed, hedgehog,
and dpp. What determines the fate of a cell in the wing A/P axis?
- What is the difference between serial induction
(signal relay) and direct long range action of a secreted signal?
Direct long range action of dpp is supported by
Clones of cells mutant for the dpp receptor that are located far away from
the cells expressing dpp are not influenced by the dpp signal. If the action
of dpp on these cells was indirect (i.e. through a relay of signals other
than dpp), the loss of the dpp receptor in these cells would have no effect.
The expression of marker genes induced by the dpp signal is restricted to
clones of cells that express an ligand independent, activated form of the
dpp receptor. If a signal relay were occurring, expression of marker genes
expressed distant from the source of dpp would be expected to be induced
outside the clone (see Lecuit et al., 1996; Nellen et al.,
1996 for evidence that the action of dpp is direct).
Thus, the secretion of dpp by cells in a stripe in the centre of
the disc may set up a concentration gradient of dpp across the wing
imaginal disc. This has been shown to establish broad domains of gene expression
in a manner that may be analogous to the way the gap gene domains are established
in the embryo in response to the bicoid gradient.
Reviews* and References
Basler, K., and Struhl, G. (1994). Compartment
boundaries and the control of Drosophila limb pattern by hedgehog protein.
Nature 368, 208-14.
*Blair, S. S. (1995). Compartments and appendage development in Drosophila.
Bioessays 17, 299-309.
*Brook, W. J., Diaz-Benjumea, F. J., and Cohen, S. M. (1996). Organizing
spatial pattern in limb development. Ann Rev Cell Dev Biol 12, 161-180.
Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental
Biology. Third edition. Saunders College Pub. Philadelphia.
Cohen, S. M. (1993). Imaginal disc development. In
Drosophila Development, A. Martinez-Arias and M. Bate, eds. (Cold Spring
Harbor: Cold Spring Harbor Press), pp. 747-841.
Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer.
Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill.
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H., and Cohen, S. M.
(1996). Two distinct mechanisms for long-range patterning by Decapentaplegic
in the Drosophila wing. Nature 381, 387-93.
Nellen, D., Burke, R., Struhl, G., and Basler, K. (1996). Direct and long-range
action of a DPP morphogen gradient. Cell 85, 357-68.
Tabata, T., Schwartz, C., Gustavson, E., Ali, Z., and Kornberg, T. B. (1995).
Creating a Drosophila wing de novo, the role of engrailed, and the compartment
border hypothesis. Development 121, 3359-69.
Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and
Meyerowitz, E. 1998. Principles of Development. Current Biology.
Zecca, M., Basler, K., and Struhl, G. (1995). Sequential organizing activities
of engrailed hedgehog and decapentaplegic in the Drosophila wing. Development